The invention relates to mutationally altered monoclonal antibodies, methods of producing mutationally altered monoclonal antibodies, recombinant polynucleotides encoding mutationally altered immunoglobulins, methods for site-directed mutation of immunoglobulin coding sequences that alter post-translational glycosylation of immunoglobulin polypeptides, expression vectors and homologous recombination vectors for constructing and expressing mutationally altered immunoglobulins, and cells and animals that express mutationally altered immunoglobulins.
Glycosylation of immunoglobulins has been shown to have significant effects on their effector functions, structural stability, and rate of secretion from antibody-producing cells (Leatherbarrow et al., Mol. Immunol. 22:407 (1985)). The carbohydrate groups responsible for these properties are generally attached to the constant (C) regions of the antibodies. For example, glycosylation of IgG at asparagine 297 in the CH2 domain is required for full capacity of IgG to activate the classical pathway of complement-dependent cytolysis (Tao and Morrison, J. Immunol. 143:2595 (1989)). Glycosylation of IgM at asparagine 402 in the CH3 domain is necessary for proper assembly and cytolytic activity of the antibody (Muraoka and Shulman, J. Immunol. 142:695 (1989)). Removal of glycosylation sites as positions 162 and 419 in the CH1 and CH3 domains of an IgA antibody led to intracellular degradation and at least 90% inhibition of secretion (Taylor and Wall, Mol. Cell. Biol. 8:4197 (1988)).
Glycosylation of immunoglobulins in the variable (V) region has also been observed. Sox and Hood, Proc. Natl. Acad. Sci. USA 66:975 (1970), reported that about 20% of human antibodies are glycosylated in the V region. Glycosylation of the V domain is believed to arise from fortuitous occurrences of the N-linked glycosylation signal Asn-Xaa-Ser/Thr in the V region sequence and has not been recognized in the art as playing an important role in immunoglobulin function.
It has been reported that glycosylation at CDR2 of the heavy chain, in the antigen binding site, of a murine antibody specific for xcex1-(1-6)dextran increases its affinity for dextran (Wallick et al., J. Exp. Med. 168:1099 (1988) and Wright et al., EMBO J. 10:2717 (1991)).
M195 is a murine IgG2a monoclonal antibody that binds CD33 antigen and has therapeutic potential for the treatment of myloid leukemia (Tanimoto et al., Leukemia 3:339 (1989) and Scheinberg et al., Leukemia 3:440 (1989)). M195 binds to early myeloid progenitor cells, some monocytes, and the cells of most myeloid leukemias, but not to the earliest hematopoietic stem cells.
The efficient cellular binding and internalization of M195 has allowed use of the radiolabeled antibody in clinical trials for acute myelogenous leukemia (AML) (Scheinberg et al., J. Clin. Oncol. 9:478 (1991)). The murine M195 antibody, however, does not kill leukemic cells by complement-dependent cytotoxicity with human complement, or by antibody-dependent cellular cytotoxicity with human effector cells. The human anti-mouse antibody (HAMA) response may also preclude long term use of the murine antibody in patients. To increase the effector function and reduce the immunogenicity of the M195 antibody in human patients, chimeric and humanized versions of the antibody have been constructed (Co et al., J. Immunol. 148: 1149, (1992)). The chimeric antibody combines the murine M195 V region with a human C region, while the humanized antibody combines the complementarity determining regions (CDRS) of murine M195 with a human antibody V region framework and C region (Co et al., op.cit.). The construction and characterization of chimeric and humanized M195 antibodies of the human IgG1 isotype is described (Co et al., op.cit.).
While the production of so called xe2x80x9cchimeric antibodiesxe2x80x9d (e.g., mouse variable regions joined to human constant regions) has proven somewhat successful in reducing the HAMA response, a significant immunogenicity problem remains. Moreover, efforts to immortalize human B-cells or generate human hybridomas capable of producing human immunoglobulins against a desired antigen have been generally unsuccessful, particularly with many important human antigens. Most recently, recombinant DNA technology has been utilized to produce immunoglobulins which have human framework regions combined with complementarity determining regions (CDR""s) from a donor mouse or rat immunoglobulin (see, e.g., EPO Publication No. 0239400). These new proteins are called xe2x80x9creshapedxe2x80x9d or xe2x80x9chumanizedxe2x80x9d immunoglobulins and the process by which the donor immunoglobulin is converted into a human-like immunoglobulin by combining its CDR""s with a human framework is called xe2x80x9chumanizationxe2x80x9d. Humanized antibodies are important because they bind to the same antigen as the original antibodies, but are less immunogenic when injected into humans.
However, a major problem with humanization procedures has been a loss of affinity for the antigen (Jones et al., Nature, 321, 522-525 (1986)), in some instances as much as 10-fold or more, especially when the antigen is a protein (Verhoeyen et al., Science, 239, 1534-1536 (1988)). Loss of any affinity is, of course, highly undesirable. At the least, it means that more of the humanized antibody will have to be injected into the patient, at higher cost and greater risk of adverse effects. Even more critically, an antibody with reduced affinity may have poorer biological functions, such as complement lysis, antibody-dependent cellular cytotoxicity, or virus neutralization. For example, the loss of affinity in the partially humanized antibody HUVHCAMP may have caused it to lose all ability to mediate complement lysis (see, Riechmann et al., Nature, 332, 323-327 (1988); Table 1).
Therefore, there exists a need in the art for immunoglobulins that have an altered affinity for antigen, particularly an increased affinity and/or increased specificity for an antigen, and, desirably, potentially lower immunogenicity and improved effector function conferred by naturally-occurring constant region glycosylation. For example, an immunoglobulin having one or more human constant region effector functions and an improved binding affinity and/or specificity characteristic of the M195 antibody variable region may eliminate the need for radiolabeling and allow repeated does in therapeutic trails. Additionally, there is a need in the art for methods that produce immunoglobulins which have improved binding affinity and/or specificity for an antigen, but which do not have significantly increased immunogenicity. Thus, there exists a need in the art for methods to increase the efficacy and reduce the required doses of immunoglobulins of therapeutic importance, and immunoglobulins produced by such methods.
This invention provides methods for producing mutated immunoglobulins, particularly mutated monoclonal antibodies that have an increased affinity and/or a modified specificity for binding an antigen, wherein the modification of the antigen binding property results from an introduction of at least one mutation in an immunoglobulin chain variable region (V region) that changes the pattern of glycosylation in the V region. Such mutations may add a novel glycosylation site in the V region, change the location of one or more V region glycosylation site(s), or preferably remove a pre-existing V region glycosylation site, more preferably removing an N-linked glycosylation site in a V region framework, and most preferably removing an N-linked glycosylation site that occurs in the heavy chain V region framework in the region spanning about amino acid residue 65 to about amino acid residue 85, using the numbering convention of Co et al. (1992) op.cit. In a preferred embodiment, the method of the invention does not substantially modify glycosylation of constant regions. A preferred method introduces V region mutations that increase the antibody affinity for specific antigen.
The present invention also provides mutant immunoglobulins with an altered antigen binding property, preferably glycosylation-reduced antibodies which have at least one V region glycosylation site removed by mutation. Preferably such mutant immunoglobulins include a mutated immunoglobulin heavy chain variable region, and more preferably include an entire mutated immunoglobulin heavy chain. In some embodiments, a mutant antibody will include at least one mutated heavy chain portion and at least one mutated light chain portion. In preferred embodiments, a mutant antibody will include at least one mutated full-length heavy chain and at least one mutated full-length light chain, wherein either or both heavy and light chain species may be naturally-occurring, chimeric, or humanized. Alternatively, in some embodiments it is preferred that mutated antibodies include a mutated heavy chain and an unmutated light chain, or vice versa.
A preferred embodiment of the invention is a mutant antibody that includes a glycosylation-reduced immunoglobulin chain, wherein at least one naturally-occurring V region glycosylation site, preferably at a position in the V region framework, has been removed by mutation. In some preferred embodiments, a glycosylation-reduced immunoglobulin chain is a heavy chain wherein at least one carbohydrate moiety is attached to a constant region amino acid residue through N-linked glycosylation.
This invention further provides sterile compositions of therapeutic immunoglobulins for treating disease in mammals, comprising a unit dosage of a mutant immunoglobulin, or a mixture of mutant immunoglobulins, having enhanced antigen binding properties.